Development and evaluation of an unlabeled probe high‐resolution melting assay for detection of ATP7B ATP7B mutations in Wilson's disease

摘要

Background Wilson's disease ( WD ) is a rare autosomal recessive disorder characterized by the deposition of copper mainly in the liver or nerve system that leads to their dysfunction. Mutations in the gene encoding ATP ase, Cu+ transporting, beta polypeptide ( ATP 7B ) are causative for WD . The aim of this study was to develop a rapid and convenient assay for detection of the three most common causative ATP 7B mutations, p.R778L, p.P992L, and p.V1106I. Methods Plasmids containing DNA fragments harboring each of the three ATP 7B mutations were constructed. High‐resolution melting ( HRM ) analysis was conducted by asymmetric polymerase chain reaction ( PCR ) amplification with paired primer and unlabeled probe, performed in a 96‐well plate formatted LightCycler 480 Real‐Time PCR System. The assay was evaluated for accuracy and reproducibility by genotyping of 41 WD cases. Results The unlabeled probe HRM assays performed on constructs with the p.R778L, p.P992L, and p.V1106I mutations in the ATP 7B gene resulted in additional melting peaks. According to the unlabeled probe HRM molecular signature, we could differentiate homozygous mutations from wild‐type with the ΔTm (difference between melting temperatures) 4°C, and the coefficient of variation in repeatability tests was 5%. In the validation assay using our method to examine clinical samples, a 100% accuracy rate was achieved. Conclusions The newly developed assay to rapidly genotype the ATP7B mutations is convenient, accurate, and reproducible, and represents a favorable alternative to Sanger sequencing in the identification of specific ATP 7B mutations.